Efficacy of variable dosage of aspirin in combating methotrexate-induced intestinal toxicity

Abstract

The aim of the present study was to study in detail the effect of variable doses of aspirin on intestinal toxicity. Albino rats were randomly divided into six groups and subjected to 13 weeks treatment against a sham control (3 ml kg⁻¹ by mouth (p.o.) normal saline); a toxic control (2.5 ml kg⁻¹ intraperitoneal injection (i.p.), MTX); a low dose of aspirin (8 mg kg⁻¹, p.o.); a low dose of aspirin plus MTX (8 mg kg⁻¹, p.o. + 2.5 ml kg⁻¹, i.p.), a high dose of aspirin (45 mg kg⁻¹, p.o.); and a high dose of aspirin plus MTX (45 mg kg⁻¹, p.o. + 2.5 ml kg⁻¹, i.p.). The intestinal toxicity of aspirin was assessed on the basis of biochemical changes and modulation in the inflammatory markers. Low doses of aspirin gave significant protection against MTX-induced toxicity, whereas high dose failed to do so. High doses of aspirin also produced adverse biochemical changes in the physiology, but low dose did not.

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1. Introduction

Methotrexate (MTX) is an anticancer agent and an effective inhibitor of folic acid production. MTX competitively inhibits dihydrofolate reductase (DHFR), an enzyme required for the synthesis of folic acid. Folic acid is required for the synthesis of purines.¹ MTX is used as an antimetabolite in cancer chemotherapy, including lymphocytic leukemia, non-Hodgkin's lymphoma, osteosarcoma, head and neck cancer, and mammary gland tumors.² In addition, MTX is also prescribed for the treatment of auxiliary disease conditions such as rheumatoid arthritis (RA) and refractory inflammatory bowel disease.³ MTX inhibits the remethylation of homocysteine, a process that forms reactive oxygen species (ROS) such as superoxide and hydrogen peroxide.⁴

MTX-associated chemotherapy may directly induce mucositis by causing deoxy-ribonucleic acid (DNA) strand breakage, either by generating ROS or by provoking enzymatic or transcription factor in cellular elements within the mucosa. ROS may damage cells other than cancerous tissues and may also activate secondary mediators of injury, including transcription factors such as nuclear factor-κβ (NF-κβ).⁵ Activation of transcription factors in response to ROS, radiotherapy or chemotherapy results in gene up-regulation for tumor necrosis factor-α (TNF-α) and interleukins (IL-1β and IL-6).⁶ Previous studies have investigated changes in levels of NF-κβ and other pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, and also interleukin (IL)-1β and IL-6 in experimental MTX-treated animals.^7,8 This leads to tissue injury and apoptosis of cells in the submucosa and primary injury of cells within the basal epithelium, and also to mucositis.⁹ MTX-induced injury within the intestinal tissue is thus associated with hyperpolarization, and injury to the mucosal, submucosal and basal epithelium due to ROS production, and consequent regulation of pro-inflammatory cytokines and mediators.⁶

MTX is often used as an initial disease-controlling antirheumatic agent by modifying the immune system, and is commonly prescribed with other drugs, including non-steroidal anti-inflammatories (NSAIDs), like aspirin.¹⁰ In the past, there have been safety concerns about the use of painkillers, NSAIDs and aspirin, with MTX in patients with inflammatory disorders.¹¹ The simultaneous use of these drugs with MTX may increase the risk of toxicity, causing mouth ulcers, nausea, vomiting, hepatotoxicity, cardiotoxicity or bone marrow depression.^12,13 In these cases, close monitoring of the vital parameters is essential.

In view of the therapeutic advantages of the MTX– and NSAID–aspirin combination, and the risk of associated toxicities, it was considered to be worth assessing the effect of low and high doses of aspirin accompanying the administration of MTX.

2. Materials and methods

2.1 Animals

The Wistar strain of albino rats of both sexes (120–140 g) were obtained from the central animal house. Animals were kept under controlled conditions in polypropylene cages at room temperature (25 ± 2 °C) with 12 h cycles of light and dark. The animals were fed with standard laboratory animal feed and water without restriction, and were subject to experimental conditions for no more than one week. The experimental protocol was ratified by the Institutional Animal Ethics Committee (IAEC) (approval no. UIP/IAEC/2014/FEB/09).

2.2 Drugs and chemicals

MTX originated from Folitrax-15, IPCA Laboratories Limited, Mumbai, India, and was purchased from a standard commercial supplier, and aspirin was purchased from Hi-media, Mumbai, India. The colorimetric kits for serum glutamic oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) were supplied by Recombigen Laboratories Private Limited, New Delhi, and lactate dehydrogenase from Crest Bio Systems, Goa. The commercial ELISA kits for cyclo-oxygenase (COX-1 and -2) and lipoxygenase (15-LOX) came from Cayman Chemicals, Ann Arbor, MI, USA. All other chemicals were of analytical grade and purchased from Hi-media, Mumbai, India.

2.3 Experimental design

Animals were randomly selected and divided into six groups of six, and subjected to the treatment presented in Table 1. MTX (2.5 mg kg⁻¹, i.p.) was administered initially for one week and then concurrently with aspirin (8 mg kg⁻¹, p.o. and 45 mg kg⁻¹, p.o.) for thirteen weeks thereafter. Aspirin was preferentially given orally since this is the most common method of administration in humans, giving the study a practical basis. The intraperitoneal route was chosen for MTX administration, this being the standard route for establishing intestinal toxicity. After treatment for 13 weeks, blood was collected from the retro-orbital plexus. The blood samples were incubated for 1 h at 37 °C and centrifuged at 10 000 rpm to collect the serum. The animals were finally sacrificed under light ether anesthesia, and the intestinal tissues collected after securing each end with a surgical suture to avoid drainage of the contents. The serum and intestinal tissue were retained for further investigation. High and low doses of aspirin were selected on the basis of previous literature citing the anti-inflammatory and antiplatelet dosage of aspirin for use in albino rats.^14,15 Aspirin was dissolved in warm water.

Table 1 Effect of aspirin on pH, total acidity, free acidity and CMDI in MTX-induced intestinal damage^a,b,c

S no.	Group	Treatment	pH	Total acidity (mEq l^−1)	Free acidity (mEq l^−1)	CMDI
a Each group contained six animals. Values are represented as mean ± SD.b Statistical significance was compared to toxic control using one-way ANOVA followed by Bonferroni test (*p < 0.05, **p < 0.01 and ***p < 0.001).c Values in parenthesis represent percentage inhibition.
1.	Group I	Sham control (normal saline, 3.0 ml kg^−1, p.o.)	7.15 ± 0.02	10.99 ± 1.67	7.32 ± 1.96	0.17 ± 0.40
2.	Group II	Toxic control (MTX, 2.5 ml kg^−1, i.p.)	6.27 ± 0.04	14.38 ± 1.42	11.92 ± 0.92	4.00 ± 0.00
3.	Group III	Aspirin (8 mg kg^−1, p.o.)	7.67 ± 0.02***	10.52 ± 0.45*** (26.84%)	7.94 ± 0.45*** (33.38%)	0.83 ± 0.40*** (79.25%)
4.	Group IV	Aspirin + MTX (8 mg kg^−1, p.o. + 2.5 ml kg^−1, i.p.)	7.40 ± 0.02***	10.52 ± 0.43*** (26.84%)	7.70 ± 0.51*** (35.40%)	1.83 ± 0.40*** (54.25%)
5.	Group V	Aspirin (45 mg kg^−1, p.o.)	7.73 ± 0.06***	11.37 ± 0.64*** (20.93%)	8.88 ± 0.58*** (25.50%)	1.83 ± 0.40*** (54.25%)
6.	Group VI	Aspirin + MTX (45 mg kg^−1, p.o. + 2.5 ml kg^−1, i.p.)	7.50 ± 0.02***	14.47 ± 0.77 (0.76%)	11.40 ± 0.77 (4.36%)	2.83 ± 0.75** (29.25%)

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2.4 Enzymatic markers for heart and liver

The serum samples were examined for liver (SGOT and SGPT) and cardiac (LDH) marker enzymes using commercial colorimetric assay kits, following the manufacturer's protocol.

2.5 Estimation of pH, and free and total acidity

The intestinal contents were examined for intestinal pH (Hanna Instruments, HI 98107), and free and total acidity, using the procedure described previously.^16,17

2.6 Assessment of colonic mucosal disease index (CMDI)

Colonic tissue approximately 10 cm from the anus was taken, opened longitudinally, washed in normal saline buffer and fixed on a wax block. Scoring was recorded as follows: 0 = normal mucosa; 1 = mild hyperemia, no erosion or ulcers on the mucosa surface; 2 = moderate hyperemia, with erosion or ulcers on the mucosal surface; 3 = severe hyperemia, necrosis and ulcers on the mucosal surface, ulcerative area less than 40%; 4 = severe hyperemia, necrosis and ulcers on the mucosa surface, with ulcerative area above 40%.¹⁸

2.7 Biochemical estimations

The intestinal tissues were evaluated for the biochemical parameters for TBARS,¹⁹ SOD,²⁰ glutathione (GSH),²¹ catalase²² and protein carbonyl,²³ using the established methods, at our laboratory.

2.8 Inflammatory cascade enzymes

The intestinal tissues were further evaluated for the enzymatic activities of COX-1, COX-2 and 15-LOX using commercial Elisa kits and an Alere microplate reader (AM 2100, Alere Private Limited, New Delhi, India).

2.9 Morphological evaluation

Intestinal tissues from all the groups were examined for morphological changes using scanning electron microscopy. Samples were fixed in 2.5% glutaraldehyde for 6 h at 4 °C and washed in 0.1 M phosphate buffer, with three changes each of 15 min, at 4 °C. 1% osmium tetroxide was used for post-fixation for 2 h at 4 °C and samples were washed in 0.1 M phosphate buffer, with three changes each of 15 min, at 4 °C to remove unreacted fixative. Specimens were dehydrated using increasing concentrations of dry acetone (30%, 50%, 70%, 90%, 95% and 100%) at 4 °C for 30 min periods. The specimens were then air dried and mounted on an aluminium stub with adhesive tape. The tissues were inspected for morphological changes using a scanning electron microscope (JEOL-JSM-6490LV).¹⁶

2.10 Statistical analysis

All data were presented as mean ± standard deviation and analyzed by one-way ANOVA, followed by the Bonferroni test for identification of possible significance between the various groups: *p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant. Statistical analysis was carried out using Graph Pad Prism software (3.2), San Diego, CA.

3. Results

The investigation confirmed that significant protection against MTX toxicity could be provided by a low dose of aspirin, which produced a reduction in the CMDI. Relative to the toxic control, a low dose of aspirin led to the restoration of pH, total acidity and free acidity (Table 1). When examined biochemically, MTX administration gave a conspicuous increase in the tissue malondialdehyde (MDA) level, and this was restored to normal by aspirin. Furthermore, tissue GSH levels were substantially increased in the MTX-treated group (42.95 ± 6.93) in contrast to the sham control (16.47 ± 4.47).The enzymatic activity of SOD was seen to be increased after the MTX and aspirin treatment, and the enzymatic activity of catalase fell somewhat after aspirin treatment, similar to the toxic control. When protein carbonyl was assessed in terms of protein oxidation, the antiplatelet dose of aspirin produced a significant decrease in protein oxidation, shown as a fall in protein carbonyl concentration (104.47 ± 1.35), analogous to the toxic control (116.81 ± 0.39) (Table 2). It was interesting that biochemically a low dose of aspirin exhibited better protection than a high dose.

Table 2 Effect of aspirin on TBARS, GSH, SOD, catalase and protein carbonyl in MTX-induced intestinal toxicity^a,b

S no.	Group	TBARS (nm of MDA per mg of protein)	GSH (mg %, 1 × 10^−4)	SOD (unit of SOD per mg of protein)	Catalase (nm at H2O2 per min per mg of protein)	Protein carbonyl (nm ml^−1)
a Each group contained six animals. Values are represented as mean ± standard deviation.b Statistical significance is compared to toxic control using one-way ANOVA followed by Bonferroni test. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.
1	Group I	0.71 ± 0.04	22.40 ± 0.55	16.47 ± 4.47	7.70 ± 0.81	50.45 ± 0.39
2	Group II	2.91 ± 0.02	113.32 ± 0.01	42.95 ± 6.93	37.46 ± 1.60	116.81 ± 0.39
3	Group III	0.79 ± 0.01**	26.7 ± 2.44**	26.73 ± 6.89**	8.57 ± 1.01***	70.40 ± 1.55***
4	Group IV	1.35 ± 0.01***	61.8 ± 3.13***	48.74 ± 8.14***	23.07 ± 2.27***	104.47 ± 1.35***
5	Group V	1.25 ± 0.01***	28.2 ± 2.82***	37.21 ± 7.23***	31.11 ± 2.91***	94.76 ± 3.92***
6	Group VI	1.47 ± 0.02***	98.7 ± 4.79***	44.52 ± 9.50***	34.62 ± 7.30***	109.50 ± 2.00*

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The intestinal tissues were examined for the presence of pro-inflammatory (IL-2) and anti-inflammatory cytokines (IL-4 and IL-10), and these were seen to be increased after MTX treatment. The administration of aspirin gave an additional increase (Table 3).

Table 3 Effect of aspirin on pro-inflammatory and anti-inflammatory cytokines in MTX-induced intestinal toxicity^a,b

S no.	Group	IL-2 (pg ml^−1)	IL-4 (pg ml^−1)	IL-10 (pg ml^−1)
a Each group contained six animals. Values are represented as mean ± standard deviation.b Statistical significance is compared to toxic control using one-way ANOVA followed by Bonferroni test: compare all pairs of columns; *p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant.
1	Group I	524.80 ± 0.00	108.05 ± 5.19	82.66 ± 5.95
2	Group II	764.82 ± 115.94	238.86 ± 46.84	876.98 ± 213.07
3	Group III	526.99 ± 156.71	257.31 ± 41.94	177.82 ± 0.00***
4	Group IV	968.17 ± 133.23**	300.08 ± 49.92**	1167.60 ± 79*
5	Group V	483.01 ± 31.46*	284.28 ± 15.34	1699.10 ± 66.96**
6	Group VI	683.66 ± 70.41	223.87 ± 0.00	224.91 ± 25.92**

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In view of the cardiac and hepatotoxicity associated with MTX treatment, the cardiac and liver enzymatic markers were also assayed biochemically. The administration of MTX generated significant toxicity in the cardiac tissue, clearly evident from increased MDA generation (0.93 ± 0.01) along with raised enzymatic activity of the LDH (323.01 ± 17.17), catalase (22.02 ± 3.16), and SOD (56.77 ± 2.92) against the sham control. Administration of low dose aspirin produced some restoration of MDA generation and LDH activity. An insignificant reduction in the enzymatic activities of catalase and SOD was seen after aspirin treatment (Table 4).

Table 4 Effect of aspirin on LDH, TBARS, catalase and SOD in MTX-induced toxicity^a,b

S no.	Group	LDH (unit per litre)	TBARS (nm of MDA per mg of protein)	Catalase (nm at H2O2 per min per mg of protein)	SOD (unit of SOD per mg of protein)
a Each group contained six animals. Values are represented as mean ± standard deviation.b Statistical significance compared to toxic control using one-way ANOVA followed by Bonferroni test; *p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant.
1	Group I	226.66 ± 11.44	0.48 ± 0.03	5.27 ± 0.36	19.16 ± 8.67
2	Group II	311.66 ± 17.17	0.93 ± 0.01	22.02 ± 3.16	56.77 ± 2.92
3	Group III	239.80 ± 4.24*	0.55 ± 0.01**	4.84 ± 0.51**	25.92 ± 0.41***
4	Group IV	307.21 ± 0.57**	0.79 ± 0.02***	19.46 ± 2.41	53.98 ± 2.21
5	Group V	266.85 ± 4.97	0.62 ± 0.01**	7.16 ± 4.02***	33.93 ± 12.11***
6	Group VI	403.30 ± 9.67**	0.86 ± 0.03**	29.75 ± 10.07	56.87 ± 2.76

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When assessed on the basis of the hepatic enzymes, MTX was found to be hepatotoxic, with increased levels of SGOT (44.50 ± 1.95) and SGPT (43.96 ± 0.88) in comparison to the control. Treatment with low doses of aspirin allowed an increased level of SGOT, and successively higher doses were seen to further increase enzymatic levels. In addition, aspirin at both low and high dosage also increased the SGPT levels (Table 5). Scanning electron microscopy of the intestinal tissue in the control group gave the clear impression of an overall villous and mucous pattern, and treatment with MTX produced hyper-polarization, loss of mucus and a distorted villous structure (Fig. 1).

Table 5 Effect of aspirin on liver marker enzymes in MTX induced toxicity^a,b

S. no.	Group	SGOT (unit dl^−1)	SGPT (unit dl^−1)
a Each group contains six animals. Values are represented as mean ± SD.b Statistical significance compared to toxic control using one-way ANOVA followed by Bonferroni test: compare all pairs of columns,*p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.
1	Group-I	23.34 ± 0.20	20.88 ± 1.51
2	Group-II	44.50 ± 1.95	43.96 ± 0.88
3	Group-III	24.69 ± 1.01***	30.49 ± 0.64**
4	Group-IV	30.38 ± 1.25**	52.65 ± 0.98**
5	Group-V	40.62 ± 0.66**	36.21 ± 3.77**
6	Group-VI	58.70 ± 1.80***	65.73 ± 1.79***

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Fig. 1 Photomicrographs of the scanning electron microscopy analysis in the treatment groups. (A) Control (3 mg kg⁻¹ p.o., normal saline) (B) toxic control (MTX, 2.5 mg kg⁻¹, i.p.) (C) aspirin (8 mg kg⁻¹, p.o.) (D) aspirin (8 mg kg⁻¹, p.o.) + MTX (2.5 mg kg⁻¹, i.p.) (E) aspirin (45 mg kg⁻¹, p.o.) (F) aspirin (45 mg kg⁻¹, p.o.) + MTX (2.5 mg kg⁻¹, i.p.).

4. Discussion

MTX is extensively used as a chemotherapeutic agent of the antimetabolite type, frequently associated with side-effects such as intestinal toxicity.²⁴ In recent years, the use of MTX has been extended from that of an exclusively chemotherapeutic agent to include use as an antirheumatic and antipsoriatic agent. The extended therapeutic profile of MTX has unfortunately been associated with toxicities such as cardiotoxicity, nephrotoxicity and hepatotoxicity.^4,25,26 However, one of its most important and extensively discussed toxic effects has been intestinal damage, causing malabsorption and diarrhoea, resulting in severe weight loss, and in turn, upsetting the therapeutic regimen.²⁷ MTX-induced toxicities have included inflammation, due to the generation of pro-inflammatory cytokines, and alterations associated with the antioxidant defense mechanism through the generation of ROS.⁶ The present study was designed to investigate the effect of aspirin as an anti-inflammatory agent against MTX toxicity. Bearing in mind the gastrointestinal side-effects associated with anti-inflammatory (high) doses of aspirin, it was decided to include anti-platelet (low) doses in addition.

Treatment with low doses of aspirin was seen to give protection against intestinal toxicity when observed through physiological parameters. The aspirin treatment gave a significant increase in intestinal pH, with a decrease in both total and free acidity. The CMDI score in the toxic control was also significantly reduced by aspirin in a dose-dependent manner; indeed aspirin gave marked protection against MTX-induced intestinal toxicity.

Diversification in the antioxidant defense mechanisms in the intestinal tissue associated with MTX treatment has been extensively reported and is an accepted mechanism of MTX toxicity. The study in fact showed increased MDA production in the MTX-treated group.

MDA is a product of phospholipid peroxidation and is a marker for oxidative stress. The regimen with aspirin reduced MDA production to a reasonable level. It was significant that an antiplatelet (low) dose of aspirin was more appropriate than an anti-inflammatory (high) dose.

Glutathione (GSH) plays a dominant role in insulating tissues from peroxidative attack, and MTX significantly increased the level of tissue GSH, contradicting earlier findings suggesting depletion of GSH in tissues during oxidative stress.²⁸ The increased GSH levels observed in our study might possibly have been the result of increased biogenesis of GSH due to oxidative stress, the product of a feedback mechanism. The administration of aspirin with MTX gave a significant decrease in tissue GSH levels, and this could have been due either to the restored biogenesis of GSH or to diminished levels of oxidative stress as a result of aspirin therapy.^29,30

The combined effect of SOD and catalase provides a major defense against ROS. SOD forms hydrogen peroxide through its scavenging action on the superoxide radical, and this scavenging effect is further assisted by catalase (a haem protein), which catalyzes the breakdown of hydrogen peroxide to H₂O and molecular oxygen, thus protecting the tissue from highly reactive OH⁻ radicals. We observed increased enzymatic activity of SOD and catalase, in disagreement with previous studies, which have mostly observed for decreased levels of SOD, and subsequently catalase, as a consequence of oxidative stress.³¹ In this study, we were open also to the possibility of increased activity of both SOD and catalase. One could foresee a diminished activity of SOD and catalase as a result of an amelioration of oxidative stress, but we considered that physiological compensatory mechanisms for dealing with oxidative stress could be a possible reason for our observations.

A simultaneous increase in catalase activity is essential in order to outweigh the superoxide scavenging caused by SOD, and this was observed in our study. ROS can damage all types of biological molecules, in addition to lipids, DNA and proteins. The alterations to proteins directly caused by oxidative stress on amino acid residues can be linked to the disposition of carbonyl derivatives, a widely used marker for protein oxidation.³² Studying the formation of protein carbonyls has an advantage over lipid peroxidation, as oxidized proteins are more substantial and the protein carbonyl is therefore universally selected as a marker of oxidative stress.²³ In the present study, MTX produced a noticeable increase in protein carbonyl content, denoting protein oxidation. Aspirin at antiplatelet dose level produced a substantial decrease in protein carbonyl content in comparison to MTX-treated groups, and aspirin normalised the levels of physiological antioxidant defence. The antiplatelet dose of aspirin produced a more effective response towards MTX toxicity than a high dose.

To give further insight, we examined the role of pro- and anti-inflammatory cytokines in intestinal tissues, and found a significant increase in IL-2, IL-4 and IL-10 levels following administration of MTX.

IL-2 is a signalling molecule in immune systems and oversees the activities of the leukocytes and lymphocytes responsible for immunity. An increased level of IL-2 expression was revealed after MTX treatment, and in addition aspirin (low dose) increased the level of IL-2 when administered in conjunction with MTX. On the other hand, a higher dose of aspirin produced an insignificant decrease in the IL-2 level, due to the simple fact that this was an anti-inflammatory dose. This finding did not agree with previous studies, which found decreased levels of IL-2 in the serum of patients, including rheumatoid patients, treated with MTX.³³

A previous study has shown that ex vivo treatment of peripheral blood monocytes with MTX increased the expression of IL-4 and IL-10, which is in line with the present study. However, aspirin did not produce any lowering of IL-4 levels, either at high or low dose. IL-10 is an anti-inflammatory cytokine and underwent a striking increase following the MTX treatment, adopting an immuno-compromised status due to its immuno-regulatory properties. A high dose of aspirin significantly reduced the levels of IL-10, whereas the low dose produced increased IL-10 levels. In addition to the other dominant toxicities, MTX has been noted both clinically and preclinically to be cardiotoxic and hepatotoxic.³⁴ Taking this into account and to gain a better understanding, we looked at serum SGOT and SGPT levels. The results demonstrated greatly increased hepatotoxicity, accompanied by a large increase in serum SGOT and SGPT levels. Treatment with low doses of aspirin produced considerable protection by normalizing the serum SGOT and SGPT levels. Similarly, cardiac toxicity was conspicuous through increased enzymatic activity of LDH, SOD and catalase, along with noticeable embodiment of MDA products after MTX treatment. Aspirin, at low doses, gave protection against MTX-induced cardiotoxicity. This could be credited to blockage of the COX enzyme through the acetylation of serine-529 residue. The COX-1 inhibition in the platelets leads to inhibition of TXA₂ production, which is a key platelet aggregator. Moreover, in endothelial cells COX-1 accelerates the formation of prostacyclin, which is a vital vasodilator. The dynamic balance of TXA₂ and prostacyclin modulated by aspirin provides the required balance.³⁵

When observed microscopically, the administration of MTX demonstrated significant hyperproliferation, loss of mucus and distorted villous structure, which is in concordance with earlier reports.^16,36 In comparison to MTX or aspirin (high dose), a low dose of aspirin gave significant protection either alone or in combination. The results of microscopy in the present investigation suggested that a low aspirin dose offered an advantage over high doses in combating intestinal toxicity induced by MTX.

From the foregoing investigation, one can deduce that a low dose of aspirin can provide a significant counter against MTX-induced intestinal toxicity, cardiotoxicity and hepatotoxicity. The quoted effect of low dosage aspirin could be due to its antiplatelet action, leading to impaired production of thromboxane, which binds with platelets to form a patch over damaged blood vessels. The patch may eventually become too large and block the blood flow, both locally and downstream, which is nowadays implicated against the clinical use of aspirin.³⁷ On the contrary, a high dose can advance the toxicity; in fact a low dose of aspirin can be considered a complementary therapy in MTX treatment. The use of aspirin with MTX may offer advantages, as the renal clearance of MTX or its metabolite 7-hydroxyl-MTX is not clinically hindered by aspirin.^38,39 In future, the combination of a low dose of aspirin with MTX could offer a better pharmacological profile, with less toxicity. However, the use of aspirin in combination with MTX in the clinical management of rheumatoid arthritis (RA) is in question and needs to be fully investigated.

From the above evidences, we can conclude that a low dose of aspirin can be considered as an adjuvant therapy with MTX chemotherapy and other treatment regimens, which may then reduce toxicity. However, the suitability of MTX and aspirin in the clinical management of RA is open to question. Further studies will be needed to confirm the appropriateness of the use of aspirin as an adjuvant to MTX in the clinical sphere.

Acknowledgements

The authors wish to acknowledge the assistance of Mr Mukesh Kumar, University Science Instrumentation Center (USIC), in providing SEM analysis.

References

1. S. Padmanabhan, D. N. Tripathi, A. Vikram, P. Ramarao and G. B. Jena, Methotrexate induced cytotoxicity and genotoxicity in germ cells of mice: Intervention of folic and folinic acid, Mutat. Res., 2009, 639, 43–52.
2. J. Jacques and H. Cowan Kenneth, The pharmacology and clinical use of MTX, N. Engl. J. Med., 1983, 309, 1094–1104.
3. H. S. Te, T. D. Schiano, S. F. Kuan, S. B. Hanauer, H. S. Conjeevaram and A. L. Baker, Hepatic effects of long term MTX use in the treatment of inflammatory bowel disease, Am. J. Gastroenterol., 2000, 95, 3150–3156.
4. A. P. Verdia, F. Angulo, F. L. Hardwicke and K. M. Nugent, Cardiac Toxicity Associated With High-Dose Intravenous Methotrexate Therapy: Case Report and Review of the Literature, J. Invasive Cardiol., 2005, 25, 1271–1276.
5. S. Beutheu Youmba, L. Belmonte, L. Galas, N. Boukhettala, C. Bôle-Feysot, P. Déchelotte and M. Coëffier, Methotrexate modulates tight junctions through NF-κB, MEK, and JNK pathways, J. Pediatr. Gastroenterol. Nutr., 2012, 54(4), 463–470.
6. S. T. Sonis, L. S. Elting, D. Keefe, D. E. Peterson, M. Schubert, M. Hauer-Jensen, B. N. Bekele, J. Raber-Durlacher, J. P. Donnelly and E. B. Rubenstein, Perspectives on cancer therapy-induced mucosal injury: pathogenesis, measurement, epidemiology, and consequences for patients, Cancer, 2004, 100, 1995–2025.
7. S. Kumar, M. Singh, J. K. Rawat, S. Gautam, S. A. Saraf and G. Kaithwas, Effect of Rutin against gastric esophageal reflux in experimental animals, Toxicol. Mech. Methods, 2014, 24(9), 666–671.
8. P. Raj, M. Singh, J. K. Rawat, S. Gautam, S. A. Saraf and G. Kaithwas, Effect of enteral administration of α-linolenic acid and linoleic acid against methotrexate induced intestinal toxicity in albino rats, RSC Adv., 2014, 4, 60397–60403.
9. R. M. Logan, A. M. Stringer, J. M. Bowen, R. J. Gibson, S. T. Sonisand Dorothy and M. K. Keefe, Serum levels of NF-κB and pro-inflammatory cytokines following administration of mucotoxic drugs, Cancer Biol. Ther., 2008, 7, 1139–1145.
10. A. N. Colebatch, J. L. Marks and C. J. Edward, Safety of non-steroidal anti-inflammatory drugs, including aspirin and paracetamol (acetaminophen) in people receiving MTX in inflammatory arthritis (rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, other spandyloarthritis), Cochrane Database Syst. Rev., 2011, 11, CD008872.
11. N. C. Alexander, L. M. Jonathan, M. V. Desiree and J. E. Christopher, Safety of Non-Steriodal Antiinflammatory Drugs (NSAIDs) and/or Paracetamol in people receiving Methotrexate for inflammatort arthritis: A Cochrane systemic review, J. Rheumatol., 2012, 90, 62–73.
12. J. M. Kremer, G. S. Alarcon, R. W. Lightfoot, R. F. Willkens, D. E. Furst, H. J. Williams, P. B. Dent and M. E. Weinblatt, Methotrexate for rheumatoid arthritis, suggested guidelines for monitoring liver toxicity, Arthritis Rheum., 1997, 37, 316–328.
13. K. Visser and D. M. Van der Heijde, Risk and management of liver toxicity during MTX treatment in rheumatoid and psoriatic arthritis: a systematic review of literature, Clin. Exp. Rheumatol., 2009, 27, 1017–1025.
14. N. Brunello, C. G. Albonis, C. Benatti, J. M. Blom, F. Tascedda, P. Kriwin and J. Mendlewicz, Acetyl salicylic acid accelerates and antidepressant effect of Fluoxetine in the chronic escape deficit model of depression, Int. Clin. Psycopharmacol., 2006, 21(4), 219–225.
15. M. Noa, R. Mas and C. Lariot, Protective effect of Policosanol on endothelium and intimal thickness induced by forceps in rabbits, J. Med. Food, 2007, 10(3), 452–459.
16. K. Shukla, P. Raj, A. Kumar, M. Kumar and G. Kaithwas, Effect of Monotherapy and Combination Therapy of Pantoprazole and Aprepitant in Gastric Esophageal Reflux Disease in Albino Rats, Sci. World J., 2014, 183147 , 1–7.
17. P. Khinchi, S. Saha, S. A. Saraf and G. Kaithwas, Combination therapy of gamma-aminobutyric acid derivative promotes proton pump inhibitors based healing of reflux esophagitis in animal model, Pharmacol. Rep., 2014, 66(1), 165–168.
18. M. A. Patel, P. K. Patel and M. B. Patel, Effect of ethanol extract of Ficus bengalensis (bark) on inflammatory bowel disease, Indian J. Pharmacol., 2010, 42(4), 214–218.
19. H. Ohkawa, N. Ohishi and K. Yagi, Assay of lipid peroxide in animal tissue by thiobarbituric acid reaction, Anal. Biochem., 1979, 95, 351–358.
20. S. Marklund and J. Marklund, Involvement of super oxide anion radical in the autoxidation of pyrogallol and a convenient assay of super oxide dismutase, Eur. J. Biochem., 1974, 47, 469–474.
21. G. L. Ellaman, Tissue sulphydryl group, Arch. Biochem. Biophys., 1995, 82, 70–77.
22. A. Claiborne, Catalase activity, in CRC Handbook of methods for oxygen radical research, ed. R. A. Greenwald, CRC Press, Boca Raton, 1985, pp. 283–284.
23. I. Dalle-Donne, R. Ranieri, D. Giustarini, A. Milzani and R. Colombo, Protein Carbonyl groups as biomarkers of oxidative stress, Clin. Chim. Acta, 2003, 329, 23–38.
24. V. K. Kolli, P. Abraham and B. Isaac, Alteration in antioxidant defense mechanisms in the small intestines of methotrexate treated rat may contribute to its gastrointestinal toxicity, Cancer Ther., 2007, 5, 501–510.
25. B. C. Widemann, F. M. Balis, B. Kempf-Bielack, S. Bielack, C. B. Pratt, S. Ferrari, G. Bacci, A. W. Craft and P. C. Adamson, High dose MTX- induced Nephrotoxicity in patients with osteosarcoma, Cancer, 2004, 100, 2022–2032.
26. G. Avasthi, P. Bhaat and J. Singh, Methotrexate induced liver cirrhosis in a patient of psoriasis, J. Assoc. Physicians India, 2012, 60, 47–48.
27. G. G. Altmann, Changes in the mucosa of the small intestine following methotrexate administration or abdominal X-irradiation, Am. J. Anat., 1974, 140, 263–280.
28. G. Kaithwas, P. Singh and D. Bhatia, Evaluation of in vitro and in vivo antioxidant potential of polysaccharides from Aloe vera (Aloe barbadensis Miller) gel, Drug Chem. Toxicol., 2014, 37(2), 135–143.
29. K. Gaurav and D. K. Majumdar, In vitro antioxidant and in vivo antidiabetic, antihyperlipidemic activity of linseed oil against streptozotocin induced toxicity in albino rats, Eur. J. Lipid Sci. Technol., 2012, 114, 1237–1245.
30. A. Can, A. Nuriye, N. Ozsoy, S. Bolkent, B. Pelin Arda, R. Yanrdag and A. Okyar, Effect of Aloe vera leaf gel and pulp extracts on the liver in Type-II Diabetic Rat model, Biol. Pharm. Bull., 2004, 27(5), 694–698.
31. G. Singh, A. T. Singh, A. J. Abraham, B. Bhat, A. Mukherje, R. Verma, S. K. Agarwal, S. Jha, R. Mukherjee and A. C. Burman, Protective effects of Terminalia arjuna against Doxorubicin- induced Cardiotoxicity, J. Ethnopharmacol., 2008, 117, 123–129.
32. S. Barlett Barbara and R. Stadtman Earl, Protein oxidation in Aging, Disease and oxidative stress, J. Biol. Chem., 1997, 272, 20313–20316.
33. J. Kjeldsen- Kragh, M. Hvatum and H. Scott, Antibodies against dietary antigens in rheumatoid arthritis patients treated with fasting and a one year vegetarian diet, Clin. Exp. Rheumatol., 1995, 13(2), 167–172.
34. T. Erselcan, K. J. A. Kairemo, T. A. Wiklund, M. Hernberg, C. P. Blomqvist, J. Bergh and H. Joensuu, Subclinical cardiotoxicity following adjuvant dose- escalated FEC, high dose chemotherapy or CMF in breast cancer, Br. J. Cancer, 2000, 82(4), 777–781.
35. G. Kaithwas, K. Dubey and K. K. Pillai, Effect of Aloe vera (Aloe barbadensis Miller) gel on doxorubicin-induced myocardial oxidative stress and calcium overload in albino rats, Indian J. Exp. Biol., 2011, 49, 260–268.
36. J. R. Vane, Biomedicine back to an Aspirin a Day, Science, 2002, 296, 474–475.
37. H. D. Lewis, J. W. Davis, D. G. Archibald, W. E. Steinke, T. C. Smitherman, J. E. Doherty Je, H. W. Schnaper, M. M. Lewinter, E. Linares, J. M. Pouget, S. C. Sabharwal, E. Chesler and H. Demots, Protective Effects of Aspirin against Acute Myocardial Infarction and Death in Men with Unstable Angina, N. Engl. J. Med., 1983, 309(7), 396–403.
38. J. D. Floyd, D. T. Ngujen, R. L. Lobim, R. L. Lobins, Q. Bashir, D. C. Doll and M. C. Perry, Cardiotoxicity of cancer therapy, J. Clin. Oncol., 2005, 23(30), 7685–7769.
39. G. Borek, H. R. Jenzer, L. D. Frey and J. T. Locher, Repetitive exercise induced ventricular tachycardia in a patient with rheumatoid arthritis taking low dose methotrexate, J. Rheumatol., 1992, 19, 1004–1005.

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